The present invention relates to nine amino acid-long peptides of the major outer membrane protein (xe2x80x9cMOMPxe2x80x9d) from Chlamydia trachomatis (xe2x80x9cCtxe2x80x9d). These peptides activate human cytotoxic T-lymphocytes (xe2x80x9cCTLsxe2x80x9d).
Ct is an intracellular bacterium that is the leading cause of preventable infectious blindness (ocular trachoma) in the developing world and of sexually transmitted disease (xe2x80x9cSTDxe2x80x9d) in the United States and certain other parts of the developed world. The estimated annual incidence of Ct-caused STD is in the millions. While most Ct-caused disease can be treated with antibiotics, untreated or inadequately treated infections result in hundreds of thousands of cases of pelvic inflammatory disease each year in the United States, alone.
Adverse outcomes of pregnancy, ectopic pregnancy and tubal infertility are among the consequences of genital tract infections with Ct. Moreover, apparent clearance of infection by a given serovar (serologically distinct strain of Ct) can be followed by the infection becoming latent and prolonged or by re-infection. This is important because much Ct-caused pathology results from tissue-damaging inflammatory responses of the immune system that are triggered by repeated or prolonged exposures to the whole organism. Therefore, there is a need for improved means to prevent primary infections.
A great deal of effort has been put into developing a vaccine against diseases caused by Ct infections. While whole inactivated organisms are often used as a vaccine to immunize humans, such a vaccine is not desirable in the case of Ct because certain proteins expressed by Ct, such as chlamydial heat shock proteins, induce pathological immune responses rather than protective immune responses and, thus, contribute to disease. As a result, much vaccine-related activity in chlamydial research is centered on developing a xe2x80x9csubunit vaccinexe2x80x9d that consists only of Ct protein antigens or specific parts of the proteins that elicit protective immune responses in vaccinees. The fact that B-cell responses (neutralizing antibody) to Ct MOMP protect mice from Ct-caused disease has led to a prevailing theory that MOMP, when used to vaccinate humans, might also induce protective B- and T-cell responses.
However, using whole MOMP as a vaccine is not a good solution. Whole MOMP is too difficult to isolate from natural Ct cultures in large quantities that are sufficiently pure for use in mass vaccination. Larger quantities of recombinant MOMP could theoretically be produced in E. coli, but the chemical properties (e.g. insolubility except in detergents) impede its large scale preparation as a non-toxic vaccine. Furthermore, use of whole MOMP has too much risk of adverse side effects.
Consequently, emphasis has been given to developing a subunit vaccine that contains multiple B- and T-cell xe2x80x9cepitopesxe2x80x9d in MOMP, i.e. short antigenic MOMP peptides that are recognized by B and T cells. To achieve that goal, it is critical to identify which MOMP peptides are recognized by B and T cells of infected people. To date, there have been a number of reports regarding attempts to develop vaccines based on single or multiple MOMP peptide fragments, where the focus is on raising Thxe2x80x94cell and/or B-cell responses (mostly in mice, but in some cases, in humans). See H. Su et al., 172 J. Exp. Med. 203-212 (1990) (serovar A); J. Allen et al., 147 J. Immunol. 674-679 (1991) (serovar B); M. Ishizaki et al., 60 Infect. and Immun. 3714-3718 (1992) (serovars B, C); G. Zhong et al., 151 J. Immunol. 3728-3736 (1993) (serovar B). L. Ortiz et al. 157 J. Immunol. 4554-4567 (1996) (serovar E) and U.S. Pat. No. 6,001,372 (serovar E). The disclosure of these publications and of all other publications referred to herein are incorporated by reference as if fully set forth herein.
The 371 amino acid sequence of mature MOMP of Ct serovar E, a common cause of genital tract infections, is also disclosed in L. Ortiz et al. 157 J. Immunol. 4554-4567 (1996). The naturally occurring DNA coding sequence of serovar E-MOMP is disclosed in E. Peterson et al., 18 Nuc. Acids. Res. 3414 (1990) (SEQ ID NO:9). MOMP is a transmembrane protein and comprises more than 60% of all outer membrane proteins of Ct.
Sequence analysis of MOMP from various sources has revealed that differences in amino acid sequence confined to surface-exposed xe2x80x9cvariable segmentsxe2x80x9d (VSs) of MOMP account for the serological specificity of different serovars and, also, for differences in Th-cell responses to different isolates. See M. Ishizaki et al., 60 Infect. and Immun. 3714-3718 (1992). On the other hand, membrane-embedded regions of MOMP contain amino acid sequences that are conserved among different Ct serovars, and hence the name, xe2x80x9cconstant segments (CSs)xe2x80x9d. Notably, a majority of Th-cell epitopes is located in MOMP CSs in contrast to B-cell epitopes, which are exclusively located in MOMP VSs.
T cells recognize their peptide epitopes only when they are presented on the surface of other cells in association with a particular kind of HLA (human leukocyte antigen; human MHC (major histocompatibility complex)) molecules. Different kinds of HLA molecules present different peptide epitopes. A complicating matter in vaccination of humans is the fact that HLA genes are extremely polymorphic in the human population. That is, different individuals express different HLA types, and a T-cell epitope that elicits immune responses in some individuals may not do so in others. This is a particularly troubling problem for those seeking to develop vaccines for the human population in general. Consequently, there remains a need to identify many different peptide epitopes presented by diverse HLA allotypes that elicit immune responses in the majority of population. Such a set of epitopes can then be used to create a xe2x80x9ccocktailxe2x80x9d type sub-unit vaccine containing multiple T-cell epitopes as well as B-cell epitopes.
Recently there has been a description in M. Holland et al., 107 Clin. Exp. Immunol. 44-49 (1997) of two MOMP peptides that stimulated limited CTL responses in HLA-B8+ or HLA-B35+ individuals who had experienced trachoma, an eye infection with Ct. However, only two of twelve HLA-B8+ subjects responded to the peptide therefor, and only one of thirteen HLA-B35+ subjects responded to the peptide therefor. These CTLs showed low lytic activity against targets incubated with the peptides and ability of the CTLs to lyse Ct-infected target cells was not examined.
Apart from vaccine utility, it is desirable to find CTL epitopes that can be used as components of diagnostic tests (e.g. to confirm the presence of the disease once a positive test result has been obtained using conventional tests).
In summary, the identification of human CTL epitopes is needed to design a sub-unit vaccine, and is of interest in developing diagnostic tests.
Our invention concerns a specific type of T cell responses, i.e. cytotoxic T lymphocyte (CTL) responses, in human genital tract infections with Ct. CTLs have been well documented as critical players in providing protection against infections with intracellular pathogens, including viruses, bacteria, fungi and parasites. CTLs exert their protective effector function by specifically recognizing an infected cell and secreting cytotoxic molecules that lead to the lysis and death of the infected cell, as well as the pathogens residing inside the cell. CTL recognition of an infected cell requires presentation on the cell surface of short peptide epitopes derived from proteins of the pathogens in association with HLA class I molecules.
Such peptides are generated by xe2x80x9cthe antigen processing machineryxe2x80x9d of the infected cell, which includes cytosolic proteases and the transporter molecules that move the peptides into a cellular compartment where they can bind to HLA class I molecules. It should be noted that only a fraction of peptides generated by a cell are indeed capable of activating CTLs. This is because each CTL expresses at the cell surface T-cell receptors that are specific for a single kind of HLA class I-peptide complex. It is only when the T-cell receptors bind to correct HLA class I-peptide complexes displayed by an infected cell that the CTLs become activated and capable of killing the infected cell.
In one aspect the invention provides a DNA nucleotide sequence capable of expressing a peptide of 9 to 10 amino acid residues that activates cytotoxic T-lymphocytes, the peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the peptide does not have more than 10 amino acid residues.
Such nucleotide sequences are, for example, preferably those that incorporate the applicable coding portion for a fragment of the natural MOMP gene, as described in E. Peterson et al., 18 Nuc. Acids. Res. 3414 (1990). Alternative codons that express the same amino acids may also be used.
In yet another form, the invention discloses vaccine candidates containing such peptides or nucleotide sequences. The vaccines are designed to induce cytotoxic T lymphocyte (xe2x80x9cCTLxe2x80x9d) responses in humans so as to increase the capacity of humans to resist adverse diseases resulting from Chlamydia trachomatis (xe2x80x9cCtxe2x80x9d) infection.
As noted above, activation of CTLs requires specific recognition of pathogen-derived short peptides (e.g. nine and ten mers are highly preferred in our case) which have bound to specific kinds of HLA class I molecules. Moreover, the CTL epitopes can be used to detect MOMP peptide-specific CTLs in peripheral blood of infected people and thus provides a diagnostic tool.
Detection of the MOMP-specific CTLs can be accomplished in two basic ways. First, a reagent called a xe2x80x9cHLA class I tetramerxe2x80x9d incorporating the invention can be used to enumerate MOMP peptide-specific CTLs in blood samples directly upon their removal from subjects being diagnosed for infection. Second, the CTLs in such blood samples can first be activated with our invention to proliferate in vitro and then be enumerated with the use of tetramers.
If a conventional diagnostic test based on Th-cell or B-cell antigenic response provides a negative initial result, false negatives and true positives can be diagnosed by checking blood samples using direct detection or activation followed by detection of the CTLs as the indicator. Thus, the invention can provide a method for diagnosing whether a human has been infected with Ct. Moreover, since the numbers of CTLs may reflect the stage of the infection and progress of pathogenesis, enumeration of the CTLs provides a diagnostic basis for prognosis.
The basic tools are HLA class I tetramers, each kind of tetramer incorporating just one or another of the eight claimed peptides that comprise the invention in combination with the specific kind of HLA class I molecules that binds the peptide. Each kind of tetramer binds to only those CTLs that recognize and are activated by the peptide used to make that kind of tetramer. The tetramers are fluorescently tagged in a way that allows each cell that binds a tetramer to be counted with a device called a flow cytometer. Thus, creation of the powerful tools called tetramers depends on identifying peptide epitopes that are recognized by CTLs; the invention identifies eight such epitopes.
The advantages of the present invention therefore include providing:
(a) genes coding for peptides that can activate human CTLs;
(b) sub-unit vaccines based on such genes or such peptides; and
(c) diagnostic tests using such materials.
These and still other advantages of the present invention will be apparent from the description which follows. The following description is merely of the preferred embodiments. Thus, the claims should be looked to in order to understand the full scope of the invention.